Judith A. Simcox

MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity

We examined mouse models with altered adipocyte expression of mitoNEET, a protein residing in the mitochondrial outer membrane, to probe its impact on mitochondrial function and subsequent cellular responses. We found that overexpression of mitoNEET enhances lipid uptake and storage, leading to an expansion of the mass of adipose tissue. Despite the resulting massive obesity, benign aspects of adipose tissue expansion prevail, and insulin sensitivity is preserved. Mechanistically, we also found that mitoNEET inhibits mitochondrial iron transport into the matrix and, because iron is a rate-limiting component for electron transport, lowers the rate of β-oxidation. This effect is associated with a lower mitochondrial membrane potential and lower levels of reactive oxygen species–induced damage, along with increased production of adiponectin. Conversely, a reduction in mitoNEET expression enhances mitochondrial respiratory capacity through enhanced iron content in the matrix, ultimately corresponding to less weight gain on a high-fat diet. However, this reduction in mitoNEET expression also causes heightened oxidative stress and glucose intolerance. Thus, manipulation of mitochondrial function by varying mitoNEET expression markedly affects the dynamics of cellular and whole-body lipid homeostasis.

Adipocyte iron regulates adiponectin and insulin sensitivity

Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores.

Iron and Diabetes Risk

Iron overload is a risk factor for diabetes. The link between iron and diabetes was first recognized in pathologic conditions-hereditary hemochromatosis and thalassemia-but high levels of dietary iron also impart diabetes risk. Iron plays a direct and causal role in diabetes pathogenesis mediated both by β cell failure and insulin resistance. Iron also regulates metabolism in most tissues involved in fuel homeostasis, with the adipocyte in particular serving an iron-sensing role. The underlying molecular mechanisms mediating these effects are numerous and incompletely understood but include oxidant stress and modulation of adipokines and intracellular signal transduction pathways.

Adipocyte iron regulates leptin and food intake

Dietary iron supplementation is associated with increased appetite. Here, we investigated the effect of iron on the hormone leptin, which regulates food intake and energy homeostasis. Serum ferritin was negatively associated with serum leptin in a cohort of patients with metabolic syndrome. Moreover, the same inverse correlation was observed in mice fed a high-iron diet. Adipocyte-specific loss of the iron exporter ferroportin resulted in iron loading and decreased leptin, while decreased levels of hepcidin in a murine hereditary hemochromatosis (HH) model increased adipocyte ferroportin expression, decreased adipocyte iron, and increased leptin. Treatment of 3T3-L1 adipocytes with iron decreased leptin mRNA in a dose-dependent manner. We found that iron negatively regulates leptin transcription via cAMP-responsive element binding protein activation (CREB activation) and identified 2 potential CREB-binding sites in the mouse leptin promoter region. Mutation of both sites completely blocked the effect of iron on promoter activity. ChIP analysis revealed that binding of phosphorylated CREB is enriched at these two sites in iron-treated 3T3-L1 adipocytes compared with untreated cells. Consistent with the changes in leptin, dietary iron content was also directly related to food intake, independently of weight. These findings indicate that levels of dietary iron play an important role in regulation of appetite and metabolism through CREB-dependent modulation of leptin expression.

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice

Excess iron is associated with hepatic damage and diabetes in humans, although the detailed molecular mechanisms are not known. To investigate how iron regulates glucose homeostasis, we fed C57BL/6J male mice with high‐iron (HI) diets (2 or 20 g Fe/kg chow). Mice fed an HI diet exhibited elevated AMP‐activated protein kinase (AMPK) activity and impaired insulin signaling in skeletal muscle and liver. Consistent with the increased AMPK activity, glucose uptake was enhanced in mice fed an HI diet. The effects of improved glucose tolerance induced by HI feeding were abolished in transgenic mice with expression of muscle specific dominant‐negative AMPK. Glucose output was suppressed in the liver of wild‐type mice fed an HI diet, due to decreased expression of gluconeogenic genes and decreased substrate (lactate) from peripheral glycolysis. Iron activated AMPK by increasing deacetylase and decreasing LKB1 acetylation, in turn stimulating the phosphorylation of LKB1 and AMPK. The effects of HI diet were abrogated by treatment of the mice with N‐acetyl cysteine, suggesting a redox‐dependent mechanism for increasing deacetylase activity. In addition, tissue from iron‐fed mice exhibited an elevated AMP/ATP ratio, further contributing to AMPK activation. In summary, a diet high in iron improves glucose tolerance by activating AMPK through mechanisms that include deacetylation.— Huang J., Simcox, J., Mitchell, T. C., Jones, D., Cox, J., Luo, B., Cooksey, R. C., Boros, L. G., McClain, D. A. Iron regulates glucose homeostasis in liver and muscle via AMP‐activated protein kinase in mice. FASEB J. 27, 2845–2854 (2013). www.fasebj.org

Decreased serum glucose and glycosylated hemoglobin levels in patients with Chuvash polycythemia: a role for HIF in glucose metabolism

In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel–Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia-inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzyme genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wild-type VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc, but not Pdk2, was decreased, and skeletal muscle expression of Glut1, Pdk1, and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.

Dietary Iron Controls Circadian Hepatic Glucose Metabolism through Heme Synthesis

The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling.

Global Analysis of Plasma Lipids Identifies Liver-Derived Acylcarnitines as a Fuel Source for Brown Fat Thermogenesis

Cold-induced thermogenesis is an energy-demanding process that protects endotherms against a reduction in ambient temperature. Using non-targeted liquid chromatography-mass spectrometry-based lipidomics, we identified elevated levels of plasma acylcarnitines in response to the cold. We found that the liver undergoes a metabolic switch to provide fuel for brown fat thermogenesis by producing acylcarnitines. Cold stimulates white adipocytes to release free fatty acids that activate the nuclear receptor HNF4α, which is required for acylcarnitine production in the liver and adaptive thermogenesis. Once in circulation, acylcarnitines are transported to brown adipose tissue, while uptake into white adipose tissue and liver is blocked. Finally, a bolus of L-carnitine or palmitoylcarnitine rescues the cold sensitivity seen with aging. Our data highlight an elegant mechanism whereby white adipose tissue provides long-chain fatty acids for hepatic carnitilation to generate plasma acylcarnitines as a fuel source for peripheral tissues in mice.

Mitochondrial cardiomyopathies feature increased uptake and diminished efflux of mitochondrial calcium

Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10-15day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.

Loss of TLE3 Promotes Mitochondrial Program in Beige Adipocytes and Improves Glucose Metabolism

Prolonged cold exposure stimulates the recruitment of beige adipocytes within white adipose tissue. Beige adipocytes depend on mitochondrial oxidative phosphorylation to drive thermogenesis. The transcriptional mechanisms that promote remodeling in adipose tissue are not well understood. Here we demonstrate that the transcriptional coregulator TLE3 is induced with aging and inhibits mitochondrial gene expression in beige adipocytes. Conditional deletion of TLE3 in adipocytes prevents age- and diet-induced weight gain by promoting mitochondrial oxidative metabolism and increasing energy expenditure, thereby improving glucose control. Using chromatin immunoprecipitation and deep sequencing we found that TLE3 occupies distal enhancers in proximity to nuclear-encoded mitochondrial genes and that many of these enhancers are also enriched for EBF transcription factors. TLE3 interacts with EBF2 and blocks its ability to promote the thermogenic transcriptional program. Collectively, these studies demonstrate that TLE3 mediates age-dependent beige adipose thermogenic decline through inhibition of EBF2 transcriptional activity. Inhibition of TLE3 may provide a novel therapeutic approach for obesity and diabetes.